Quality control in the secretory pathway

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1 Marek Michalak Department of Biochemistry 3-56 Medical Sciences Building Tel # Marek.Michalak@ualberta.ca Quality control in the secretory pathway Office hours: Open-door policy but to arrange a convenient time, if possible I 3 R SOC Ins 3 Agonist Nucleus ER is one of the organelles of the secretory pathway responsible for the biosynthesis, folding, assembly and modification of a large number of different soluble and membrane bound proteins. ER Ca storage, uptake and release protein synthesis, folding & posttranslational modification biosynthesis of phospholipids, cholesterol and steroids, detoxification reactions degradation of glycogen regulation of gene expression stress responses communication with mitochondria & plasma membrane` I3 R SERCA Transcription Molecular chaperones Calnexin/Calreticulin Folding factors DI Folding sensors Glucosyl transferase The quality control system prevents deployment of misofolded proteins post-translational control of protein expression ER retention for protein storage (plants) SERCA : Toyoshima et al., Nature 405:647, 2000

2 molecular chaperones - reduce the rate of protein folding - increment the efficiency in vivo calreticulin, calnexin, Bi folding enzymes - accelerate rate-limiting steps of the folding process - DI-family proteins List of ER resident chaperones and folding factors. Family Mammals Yeast Hsp90 GR94, gp96, endoplasmic Hsp70 Bi GR78 Kar2p GR170 OR150 CB-140 Lhs1p SIL1 BA Sil1p Hsp40 ERdj1 Mtj1 ERdj2 Sec63 Sec63p ERdj3 HEDJ ERdj4 ERdj5 Scj1p Jem1p Iases Cy Cy-22 Cyclophilin B Cpr5p FKB FKB2 FKB13 Fkb2p FKB7 FKB23 FKB9 FKB63 FKB10 FKB65 FKB11 FKB19 FKB14 FKB22 DI Ero and Erv family members DI DI 4HB, THB di1p,eug1p,mpd1p,mpd2p,eps1 ERp57 ERp61, ER-60 ERp72 CaB2 5 CaB1 DI DIR ERp46 C-TR, endodi ERp18 ERp19 ERp29 ERp28 ERp44 ERdj5 JDI DILT TMX1 TMX2 TMX3 DI/Erv QSOX1 Quiescin QSOX2 Quiescin-like 1 Erv Erv2p Ero Ero1α Ero1p Ero1β Lectins, glycan trimming enzymes etc. CNX/CRT Calnexin Cne1p Calreticulin Calreticulin 2 Calmegin UGGT UGGT Q9NYU2 EDEM EDEM1 Q925U4 Htm1p EDEM2 Q91VV3 EDEM3 AAH60718 Glucosidase I Gls1p Glucosidase II α subunit Gls2p Glucosidase IIβ subunit Mannosidase I ER α1,2- Mns1p How do some proteins remain in the ER whereas others move along the secretory pathway? specific retrieval signals recognized by the receptors in the CO I membranes as they form. ER Lumen soluble proteins (e.g. protein disulfide isomerase and Bi) have a retrieval signal sequence Lys-Asp-Glu-Leu or KDEL that is bound to a KDEL receptor membrane proteins (e.g. KDEL receptor) have a signal KKXX (lys-lys-?-?) at the protein C-terminal. the KKXX retrieval signal binds to CO I proteins Return to ER N-acetylglucosamine Glucose Mannose Dolichol OST N-X-S/T OST Oligosaccharyltranferase

3 G I 3 R SERCA2b C ER Lumen UD UD UD-G transferase C Ca2+ Ca Bi 2+ U ER lumen Ca2+ Gluc I Interaction with ER chaperones Ca2+ ERp57 UGGT Ca2+ UGGT ERp57 G N C DI C-Ca2+ Calcium storage CRT CNX U Grp94 U ERAD Secretion Store-operated 2+ influx SERCA structure: Toyoshima et al., Nature 405:647, 2000 Calreticulin -domain structure: Ellgaard et al., NAS 98:3133, 2001 Soluble calnexin structure: Schrag et al., Mol. Cell 8:633, 2001 Modeling of calreticulin 3D structure: Michalak et al. Cell Calcium 32:269, 2002 Calnexin Calreticulin Calnexin or Calreticulin Globular N-domain Extended arm -domain Glucose binding site Extended arm -domain Globular N-domain Glucose binding site Glucosidase II Glucosidase II C-domain Glucosidase I UD-Glc Glucosyl transferase ER Mannosidase -COO - -KDEL-COO - Glucose Mannose N-Acetyl glucosamine

4 G C-Ca2+ Ca2+ Calcium CRT storage ERp57 UGGT α1,2-mannosidase UGGT ERp57 G CNX EDEM C roperly folded glycoproteins are recognized by ERGIC53, which guides them to the cis-golgi. Misfolded proteins on the other hand are substrate to α1,2-mannosidase, which removes mannose monosaccharide units from the glycan chain of the protein. Demannosylated (misfolded) proteins are handed over from CNX to the mannose lectin EDEM. Association of the proteins with EDEM seals their fate: they are targeted for ERAD and, consequently retro-translocated to the cytosol where they are degraded by the proteasome. A model of ERp57-calreticulin complex ERp57 Extended arm -domain C A thiol-disulphide oxidoreductase also known as: as: Grp58, ER60, ERp61, ERp60, HsT17083, p58, DIA3, I-LC CRT Translocon CNX Degradation proteosome Globular N-domain Acidic C-domain -KDEL-COO- - Degradation ubiquitin Retranslocated proteins are targeted for degradation by the 26S proteasome. Retranslocation is not well understood Energy source? Receptor? Directionality? Domain structure of DI family members. Thiol-oxidase and disulfide isomerase mechanism of DI and DI-;lie proteins (including ERp57) and the pathway of electron/disulfide shuttling via Ero1 and FAD. Oxidized DI functions as disulfide donor for the oxidative folding of ER client proteins (thiol-oxidation), while reduced DI can shuffle their disulfide bonds (disulfide isomerization). For simplicity, only one of the two redox-active CXXC sites of DI is shown. DI is recharged by a cascade that consists of Ero1, its cofactor FAD and O2. Electrons flow from the ER client to the CXXC active site of DI. Next the electrons from DI flow to the CXXXXC motif of Ero1, which is present on a flexible loop and subsequently from the CXXXXC to a rigid CXXC motif present in the interior of Ero1. Finally, electrons flow from the rigid CXXC to FAD, while O2 serves as terminal electron acceptor. As a result, reactive oxygen species such as H2O2 may be generated. Signal peptides are indicated as chequered boxes; a-like domains are depicted as dark gray boxes and the CXXC(-like) motifs are indicated; inactive b-like domains as light gray boxes; calsequestrin (CSQ)-like thioredoxin domains are shown as striped gray boxes, acidic c-domains as speckled boxes; d- domains as diagonally striped boxes. The J- domain of ERdj5 as a vertically striped box and C terminal KDEL(-like) peptides that mediate ER residency in black and the tetrapeptide sequence in italic. Of the three TMX family members only TMX1 and 3 are shown, since topology of TMX2 is uncertain. Their TMDs are indicated as white boxes and their cytosolic domains as horizontally striped boxes.

5 Calreticulin deficient embryos crt +/- crt -/- Control of protein folding and calcium homeostasis in the ER are critical for many cellular functions and, when impaired, may lead to many sever pathologies d-old mouse embryos Ventricular wall What is an embryonic stem cell? EM analysis of calreticulin-deficient ventricular wall crt +/- crt -/-

6 Calreticulin is essential for cardiac differentiation of embryonic stem cells wt a-actinin MLC2v General characteristics of calnexin-deficient phenotype 30-50% smaller than wt littermates Friendly/non-aggressive ositive in acoustic startle test ositive responses to touch Good righting reflex rogressive lower limb motor disorder Gait disturbance crt -/- wt Distal muscle weakness Frequently lies on side Hunched appearance Day wt kd 21.5 crt -/- MLC2v Balance problem Ataxia Cerebellum Choroid plexus Lateral ventricular cortex Cerebral cortex CNS myelin in calnexin-deficient mice Tongue Blood vessels Aorta Cartilage of thoracic vertebral body Midgut loop Mantle layer of spinal cord Heart Liver Stomach Bladder Duodenu m Central canal of spinal cord wild-type

7 CNS myelin in calnexin-deficient mice Sciatic nerve in calnexin-deficient mice wild-type wild-type Optic nerve in calnexin-deficient mice Spine of 21-day-old mice wild-type wild-type

8 Calnexin forms transient complexes with pmp22 and 0 Lipids in the Myelin pmp pmp I anti-cnx WB anti-pmp22 or anti-0 Taken from Lancet Neurology, 2002; 1: pmp22 in calnexin-deficient cells pmp22 in calnexin-deficient cells wt wt cnx-/- ΔC CNX cnx-/- + CNX

9 Impaired protein folding results in many sever pathologies Myelin proteins arkinson s Creutzfeldt Jakob BSE prion diseases Alzheimer s Charcot Marie Tooth Disease (cmt) Atherosclerosis Cancer Huntingtons oly-q disease Myocardial Infarction Cystic Fibrosis Sickle cell Anemia α1-antitrypsin deficiency Amyotrophic lateral sclerosis (ALS) Familial amyloid polyneuropathy Gerstmann Sträussler Scheinker disease Sitosterolemia Diabetes Osteogenesis imperfecta Cataracts Light Chain Amyloidosis Lysozyme amyloidosis Downs Syndrome Fatal Familial Insomnia Kuru Gelsolin Amyloid Disease Transthyretin Amyloidogenesis Gaucher Disease Hemophilia A Loss-of-function 1α-Antitrypsin Hereditary lung emphysema β-d-galactosidase Fabry disease ABCA1 transporter Tangier disease β-glucocerebrosidase Gaucher disease β-hexoseaminidase Tay-Sachs disease β-secretase (splice variants) Alzheimer s disease Capillary morphogenesis factor-2 Infantile systemic hyalinosis Class 1 MHC heavy chain Infantile CMV-linked hepatitis CLD anion transporter Congenital chloride diarrhea Complement C1 inhibitor Hereditary angioedema Cystic fibrosis transmembrane regulator Cystic fibrosis, idiopathic pancreatitis DTDST anion transporter Diastrophic displasia Gonadotropin-releasing hormone receptor Hypogonadotropic hypogonadism Growth hormone receptor Laron syndrome Insulin receptor Diabetes mellitus, insulin-resistant syndrome Low-density lipoprotein receptor Familial hypercholesterolemia Myeloperoxidase Myeloperoxidase deficiency endrin endred syndrome olycystin-2 olycystic kidney disease 2 rotein C Venous thromboembolism Tyrosinase Oculocutaneous albinism von Willebrand factor Bleeding disorders Voltage-gated potassium channel Congenital long QT syndrome 21-Hydroxylase Congenital adrenal hyperplasia 1α-Antitrypsin β-synuclein Collagen type I IV Connexin Copper transporter Fibrillin-1 Fibrinogen Granulocyte colony-stimulating factor HERG potassium channel HMG-CoA reductase Lipoprotein lipase ael receptor Neuroligin-3 arathyroid hormone-related peptide eripheral myelin protein 22 repro-vasopressin roteolipid protein Rhodopsin Sedlin Several (presenilin, hungtingtin, r, ) TorsinA Thyroglobulin Wilson disease protein Gain-of-function Liver failure, cirrhosis arkinson s disease Osteogenesis imperfecta Charcot-Marie-Tooth syndrome Menkes disease Marfan syndrome Liver failure Severe congenital neutropenia Hereditary long QT syndrome Heart failure Familial chylomicronemia Autosomal recessive juvenile arkinsonism Asperger syndrome Hypercalcemia Charcot-Marie-Tooth syndrome Diabetes insipidus elizaeus-merzbacher leukodystrophy Autosomal dominant retinitis pigmentosa Spondylo-epiphyseal displasia tarda Neurodegenerative diseases (Alzheimer s, arkinson s, Huntington s, Creuzfeld-Jakob) Dystonia, myoclonic-dystonia syndrome Congenital hypothyroid goiter Wilson disease

10 Humans are also susceptible to several prion diseases: CJD: Creutzfeld-Jacob Disease GSS: Gerstmann-Straussler-Scheinker syndrome FFI: Fatal Familial Insomnia Kuru Alpers Syndrome These original classifications were based on a clinical evaluation of a patients family history symptoms and are still widely used, however more recent and accurate molecular diagnosis of the disease is gradually taking the place of this classification. The incidence of sporadic CJD is about 1 per million per year. GSS occurs at about 2% of the rate of CJD. It is estimated that 1 in 10,000 people are infected with CJD at the time of death. These figures are likely to be underestimates since prion diseases may be misdiagnosed as other neurological disorders. Kuru is endemic among the the Fore tribe of apua New Guinea whose rituals included eating the brain tissue of their recently deceased members of the tribe. It is characterized by headaches, joint pains and shaking of the limbs. It is believed to be caused by prions and is related to CJD. Since this practice was halted, the disease has disappeared. Before then, the disease was studied by transmitting it to chimpanzees using injections of autopsied brain tissue from human victims. It is best known for the epidemic that occurred in apua New Guinea in the middle of the twentieth century. The word kuru means "trembling with fear" in the language of the Fore people, the most commonly afflicted people. Trembling is a symptom of dying brain tissue, and as such is present in almost all patients with transmissible spongiform encephalopathy (TSE). Bovine Spongiform Encephalopathy (BSE) or "Mad Cow Disease" An epidemic of this disease began in Great Britain in 1985 and before it was controlled, over 170,000 cattle were sickened by it. Its origin appears to have been cattle feed that contained brain tissue from sheep infected with scrapie and had been treated in a new way that no longer destroyed the infectiousness of the scrapie prions. The use of such food was banned in 1988 and after peaking in 1992, the epidemic declined quickly. Alpers' disease is a rare, progressive neurodegenerative disease of the brain that occurs in infants and children. Deficiency in mitochondrial DNA polymerase gamma rsc is a protease resistant form of rc, both are extensively post translationally modified. No chemical differences between the two forms of the protein have been detected. Clearly there must be some difference. One great problem is that infectivity ratio is about 100,000:1, so infectious agent even if derived from rc may not be rsc and it could be chemically modified. A more likely possibility is that the difference between rc and rsc is conformational. rc is predominantly alpha helical and contains almost no beta sheet. The structure of rsc has not yet been determined but is predicted to be predominantly B- sheet. roposed therefore that this protein can adopt 2 quite different stable conformations. The safe rc form is normally adopted but rarely it can switch to the rsc form. Mutations favor this switch. ropose that rsc is transdominant and converts rc to rsc in an exponential fashion.